Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein

J Biol Chem. 2018 May 4;293(18):6672-6681. doi: 10.1074/jbc.RA117.000880. Epub 2018 Mar 20.


Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Keywords: Escherichia coli (E. coli); Tat system; biotechnology; maquette; protein design; protein export; protein folding; protein quality control; protein translocation; twin-arginine translocase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Circular Dichroism
  • Escherichia coli / enzymology*
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / chemistry
  • Escherichia coli Proteins / metabolism*
  • Heme-Binding Proteins
  • Hemeproteins / chemistry
  • Hemeproteins / metabolism*
  • Membrane Transport Proteins / chemistry
  • Membrane Transport Proteins / metabolism*
  • Methylamines / metabolism
  • Models, Molecular
  • Oxidoreductases, N-Demethylating / metabolism
  • Periplasm / metabolism
  • Protein Folding
  • Protein Sorting Signals
  • Protein Stability
  • Protein Transport
  • Proton Magnetic Resonance Spectroscopy
  • Substrate Specificity
  • Temperature


  • Bacterial Proteins
  • Escherichia coli Proteins
  • Heme-Binding Proteins
  • Hemeproteins
  • Membrane Transport Proteins
  • Methylamines
  • Protein Sorting Signals
  • heme protein, bacteria
  • twin-arginine translocase complex, E coli
  • Oxidoreductases, N-Demethylating
  • trimethylamine dehydrogenase
  • trimethyloxamine